Bottom Line:
Importantly, phloroglucinol decreased SLUG through inhibition of PI3K/AKT and RAS/RAF-1/ERK signaling.In agreement with in vitro data, phloroglucinol was also effective against in vivo metastasis of breast cancer cells, drastically suppressing their metastatic ability to lungs, and extending the survival time of mice.Collectively, our findings demonstrate a novel anticancer activity of phloroglucinol against metastasis of breast cancer cells, implicating its clinical relevance.

fig01: Phloroglucinol suppresses the migratory and invasive properties of breast cancer cells. (a) The chemical structure of phloroglucinol. (b, c) Dose-dependent effect of phloroglucinol on migration and invasion. Representative images are shown in (b) and migration and invasion are quantified in (c) after treatment of MDA-MB231 basal type breast cancer cells with various concentration of phloroglucinol. (d) Migration and invasion assay of BT549 basal type breast cancer cells after treatment with phloroglucinol (50 μM). (e) Effect of phloroglucinol on invasiveness of MDA-MB231 cells in 3D culture condition. Invasiveness was visualized and quantified after hematoxylin and eosin (H&E) staining. β-actin was used as a loading control. Error bars represent mean ± SD of triplicate samples. *P < 0.01 vs control.

Mentions:
To investigate whether phloroglucinol, a natural phlorotannin component of brown algae, has an anticancer activity (Fig.1a), we first treated MDA-MB231 breast cancer cells with various concentrations of phloroglucinol (0–100 μM) and analyzed its toxicity, cell growth and the effect on migration and invasion of the breast cancer cells. When cell death was analyzed by FACS after propidium iodide staining, there was no significant cell death caused by phloroglucinol in all concentrations that are tested (Fig. S1A). Importantly, however, treatment with phloroglucinol effectively suppressed the migratory and invasive ability of MDA-MB231 breast cancer cells in a dose-dependent manner (Fig.1b,c), indicating that phloroglucinol suppresses malignant phenotypes of breast cancer cells without causing cell death. Since the 50% inhibitory concentration (IC50) of phloroglucinol on migration and invasion was about 50 μM, this concentration was used for further experiments, unless otherwise mentioned. In agreement, when other basal type BT549 cells was treated with the phloroglucinol (50 μM) and analyzed the migratory and invasiveness, phloroglucinol also suppressed effectively the migratory and invasive properties of the basal type breast cancer cells in Trans-wells (Fig.1d). To confirm the effect of phloroglucinol, the invasiveness of cancer cells were also analyzed in the presence or absence of phloroglucinol by 3D-culture system, where common extracellular matrix components such as collagen I and matrigel were mixed and solidified in growth medium, mimicking in vivo tissue environment. Consistently, phloroglucinol effectively suppressed the infiltration of MDA-MB231 cells in the 3D-culture system (Fig.1e). In cell growth, treatment with phloroglucinol also caused a decrease in cell proliferation at 72 h; however, no significant increase in cell numbers was observed at 48 h after the treatment, the time point at which migration and invasion assays were performed. To consolidate this issue, MDA-MB231 and BT549 cancer cells were also treated with various concentration of phloroglucinol and the increases of cell number were analyzed at 48 h. Importantly, either concentration of phloroglucinol had no effect on breast cancer cell growth at the time point, indicating that cell growth did not affect on the accuracy of migration and invasion assays (Fig. S1B). Taken together, these results suggest that phloroglucinol has no cellular toxicity; however, it suppresses the migratory and invasive properties of breast cancer cells.

fig01: Phloroglucinol suppresses the migratory and invasive properties of breast cancer cells. (a) The chemical structure of phloroglucinol. (b, c) Dose-dependent effect of phloroglucinol on migration and invasion. Representative images are shown in (b) and migration and invasion are quantified in (c) after treatment of MDA-MB231 basal type breast cancer cells with various concentration of phloroglucinol. (d) Migration and invasion assay of BT549 basal type breast cancer cells after treatment with phloroglucinol (50 μM). (e) Effect of phloroglucinol on invasiveness of MDA-MB231 cells in 3D culture condition. Invasiveness was visualized and quantified after hematoxylin and eosin (H&E) staining. β-actin was used as a loading control. Error bars represent mean ± SD of triplicate samples. *P < 0.01 vs control.

Mentions:
To investigate whether phloroglucinol, a natural phlorotannin component of brown algae, has an anticancer activity (Fig.1a), we first treated MDA-MB231 breast cancer cells with various concentrations of phloroglucinol (0–100 μM) and analyzed its toxicity, cell growth and the effect on migration and invasion of the breast cancer cells. When cell death was analyzed by FACS after propidium iodide staining, there was no significant cell death caused by phloroglucinol in all concentrations that are tested (Fig. S1A). Importantly, however, treatment with phloroglucinol effectively suppressed the migratory and invasive ability of MDA-MB231 breast cancer cells in a dose-dependent manner (Fig.1b,c), indicating that phloroglucinol suppresses malignant phenotypes of breast cancer cells without causing cell death. Since the 50% inhibitory concentration (IC50) of phloroglucinol on migration and invasion was about 50 μM, this concentration was used for further experiments, unless otherwise mentioned. In agreement, when other basal type BT549 cells was treated with the phloroglucinol (50 μM) and analyzed the migratory and invasiveness, phloroglucinol also suppressed effectively the migratory and invasive properties of the basal type breast cancer cells in Trans-wells (Fig.1d). To confirm the effect of phloroglucinol, the invasiveness of cancer cells were also analyzed in the presence or absence of phloroglucinol by 3D-culture system, where common extracellular matrix components such as collagen I and matrigel were mixed and solidified in growth medium, mimicking in vivo tissue environment. Consistently, phloroglucinol effectively suppressed the infiltration of MDA-MB231 cells in the 3D-culture system (Fig.1e). In cell growth, treatment with phloroglucinol also caused a decrease in cell proliferation at 72 h; however, no significant increase in cell numbers was observed at 48 h after the treatment, the time point at which migration and invasion assays were performed. To consolidate this issue, MDA-MB231 and BT549 cancer cells were also treated with various concentration of phloroglucinol and the increases of cell number were analyzed at 48 h. Importantly, either concentration of phloroglucinol had no effect on breast cancer cell growth at the time point, indicating that cell growth did not affect on the accuracy of migration and invasion assays (Fig. S1B). Taken together, these results suggest that phloroglucinol has no cellular toxicity; however, it suppresses the migratory and invasive properties of breast cancer cells.

Bottom Line:
Importantly, phloroglucinol decreased SLUG through inhibition of PI3K/AKT and RAS/RAF-1/ERK signaling.In agreement with in vitro data, phloroglucinol was also effective against in vivo metastasis of breast cancer cells, drastically suppressing their metastatic ability to lungs, and extending the survival time of mice.Collectively, our findings demonstrate a novel anticancer activity of phloroglucinol against metastasis of breast cancer cells, implicating its clinical relevance.